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Super-Resolution Imaging of Live Sperm Reveals Dynamic Changes of the Actin Cytoskeleton During Acrosomal Exocytosis Ana Romarowski1, Ángel G

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Super-Resolution Imaging of Live Sperm Reveals Dynamic Changes of the Actin Cytoskeleton During Acrosomal Exocytosis Ana Romarowski1, Ángel G © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958 RESEARCH ARTICLE Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis Ana Romarowski1, Ángel G. Velasco Félix2, Paulina Torres Rodrıgueź 3,Marıá G. Gervasi4, Xinran Xu5, Guillermina M. Luque1, Gastón Contreras-Jiménez3, Claudia Sánchez-Cárdenas3,Héctor V. Ramırez-Gó ́mez3, Diego Krapf5, Pablo E. Visconti4, Dario Krapf 6, Adán Guerrero2, Alberto Darszon3 and Mariano G. Buffone1,* ABSTRACT 2015). The sperm AR occurs in mice in the upper segments of the Filamentous actin (F-actin) is a key factor in exocytosis in many cell female oviductal isthmus (Hino et al., 2016; La Spina et al., 2016; types. In mammalian sperm, acrosomal exocytosis (denoted the Muro et al., 2016) and is essential for the appropriate relocalization – acrosome reaction or AR), a special type of controlled secretion, is of proteins involved in sperm egg fusion (Satouh et al., 2012). regulated by multiple signaling pathways and the actin cytoskeleton. Acrosomal exocytosis is a highly controlled event that consists of However, the dynamic changes of the actin cytoskeleton in live sperm multiple stages that culminate in the membrane around the secretory are largely not understood. Here, we used the powerful properties of SiR- vesicle being incorporated into the plasma membrane. At the actin to examine actin dynamics in live mouse sperm at the onset of the molecular level, this complex exocytic process is regulated by AR. By using a combination of super-resolution microscopy techniques several signaling pathways involving: (1) membrane potential to image sperm loaded with SiR-actin or sperm from transgenic mice (De La Vega-Beltran et al., 2012), (2) proteins from the fusion containing Lifeact-EGFP, six regions containing F-actin within the sperm machinery system (Belmonte et al., 2016; De Blas et al., 2005), head were revealed. The proportion of sperm possessing these (3) small GTPases (Branham et al., 2009; Romarowski et al., 2015), 2+ structures changed upon capacitation. By performing live-cell imaging (4) changes in ion concentration, where Ca is a key player experiments, we report that dynamic changes of F-actin during the AR (Fukami et al., 2003; Romarowski et al., 2016a; Sánchez-Cárdenas occur in specific regions of the sperm head. While certain F-actin regions et al., 2014), and (5) changes in the actin cytoskeleton (Brener et al., undergo depolymerization prior to the initiation of the AR, others remain 2003; Romarowski et al., 2016b). In this regard, the expression of unaltered or are lost after exocytosis occurs. Our work emphasizes the actin and several actin-related proteins in mammalian sperm (some utility of live-cell nanoscopy, which will undoubtedly impact the search for of which are testis specific) suggests that actin polymerization and mechanisms that underlie basic sperm functions. depolymerization are important for sperm function (Gervasi et al., 2018; Romarowski et al., 2016b). This article has an associated First Person interview with the first author Filamentous actin (F-actin) is a key factor in exocytosis in many cell of the paper. types. It has been shown that F-actin holds together the molecular players of the secretory pathway, determines the precise site for granule KEY WORDS: Acrosomal exocytosis, Actin, Sperm exocytosis and shapes the exocytotic responses in secretory cells. In addition, stabilization of the actin network inhibits exocytosis, whereas INTRODUCTION depolymerization of this network increases the number of docked Exocytosis is the pathway by which cells selectively externalize secretory granules and enhances the exocytotic response (Chowdhury compounds and, as such, it is essential for a number of basic et al., 1999; Ehre et al., 2004; Gasman et al., 2004; Muallem et al., biological processes. In spermatozoa, acrosomal exocytosis (also 1995). As in somatic cells, the actin cytoskeleton of mammalian sperm known as acrosome reaction, denoted AR), a special type of is dynamic. While during sperm capacitation polymerization is controlled secretion, is essential for fertilization in mammals (Dan, predominant (Brener et al., 2003; Romarowski et al., 2015), during 1952). Sperm acquire the ability to undergo the AR in the female the AR, actin depolymerization occurs (Spungin et al., 1995). reproductive tract in a complex cascade of events all grouped under Previous studies regarding actin polymerization/depolymerization the name of capacitation (Puga Molina et al., 2018; Stival et al., were exclusively performed in fixed capacitated sperm or by means of using isolated membranes that had either been exposed or not to AR 1Instituto de Biologıá y Medicina Experimental (IBYME), Consejo Nacional de inducers (Brener et al., 2003; Romarowski et al., 2015). In addition, Investigaciones Cientıficaś y Técnicas (CONICET), Buenos Aires C1428ADN, most studies evaluating F-actin in mammalian sperm were performed Argentina. 2Laboratorio Nacional de Microscopıá Avanzada, Instituto de Biotecnologıa,́ Universidad Nacional Autónoma de México (UNAM), Cuernavaca, using phalloidin, which is toxic and not capable of crossing the cell Morelos 62210, México. 3Departamento de Genética del Desarrollo y Fisiologıá plasma membrane of live cells (Cooper, 1987; Vandekerckhove et al., Molecular, Instituto de Biotecnologıa,́ Universidad Nacional Autónoma de México 1985; Visegrády et al., 2004). Thus, the phalloidin staining approach is (UNAM), Cuernavaca, Morelos 62210, México. 4Department of Veterinary and Animal Science, Paige Labs, University of Massachusetts, Amherst, MA 01003, not suitable to study this dynamic process in real time using live cells. USA. 5Department of Electrical and Computer Engineering, School of Biomedical However, recent reports have demonstrated the use of a novel membrane Engineering, 1301 Campus Delivery, Fort Collins, CO 80523, USA. 6Instituto de permeable fluorescent probe, called SiR-actin, that binds to actin Biologıá Molecular y Celular de Rosario (CONICET-UNR), Rosario, Santa Fe ̌ S2000EZP, Argentina. filaments in vivo (Lukinavicius et al., 2014; Magliocca et al., 2017; Yamazaki et al., 2018). This probe allows one to image actin filaments *Author for correspondence ([email protected]) without the need to overexpress fluorescently tagged proteins, such as D.K., 0000-0002-2833-5553; M.G.B., 0000-0002-7163-6482 Lifeact, actin monomers or actin-binding proteins. In addition, SiR-actin provides a high signal-to-noise ratio, facilitating the visualization of the Received 12 April 2018; Accepted 25 September 2018 finest details of the cortical actin network and it has been successfully Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs218958. doi:10.1242/jcs.218958 visualized in neurons in stimulated emission depletion (STED) produced as a modified form of jasplakinolide, a drug that super-resolution microscopy studies (D’Este et al., 2015). is normally used to stabilize polymerized actin in vitro,itis By using this powerful new probe, we aimed to examine actin recommended to minimize the concentration of the probe and dynamics in live mouse sperm, specifically during the AR process. ideally not have a concentration of >100 nM, since higher We hypothesize that a complex and highly regulated process, such as concentrations could affect the actin cytoskeleton dynamics the AR, will be accompanied by specific changes in actin dynamics. (Lukinaviciuš et al., 2014). For this reason, we incubated sperm To this end, in live-cell imaging experiments, we set up the conditions with increasing concentrations of SiR-actin (50 to 500 nM). to simultaneously monitor actin cytoskeleton remodeling and AR Although 500 nM of SiR-actin resulted in a bright fluorescent occurrence by monitoring SiR-actin and FM4-64, respectively (Mata- signal in the sperm head and flagellum, we found that 100 nM SiR- Martínez et al., 2018; Romarowski et al., 2016a; Sánchez-Cárdenas actin (in accordance with the manufacturer’s recommendations to et al., 2014). For the first time, we report the observation of acrosomal avoid artifacts) showed a good signal-to-noise ratio (Fig. 1A). The exocytosis via super-resolution microscopy in real time. Our work signal observed using 50 nM was barely detectable. As a control, provides a detailed characterization of the F-actin cytoskeleton in cells that were not loaded with SiR-actin displayed no fluorescence the sperm head by using SiR-actin in combination with different (auto-fluorescence was not significant when excited by the far-red super-resolution microscopy approaches and transgenic mice wavelength used to detect SiR-actin; data not shown). Analysis of containing the F-actin-binding protein Lifeact-EGFP. Additionally, sperm motility by computer-assisted sperm analysis (CASA) was we observed dynamic changes of F-actin in restricted regions of the used to evaluate whether 100 nM of SiR-actin affected sperm sperm head prior to and after the initiation of the AR. Finally, our motility and viability. As shown in Fig. S1, this probe concentration work emphasizes the advantage of live-cell nanoscopy for the study did not alter either the viability or sperm motility. of ultra-structures in sperm as well as in other cell types. These F-actin is normally visualized in fixed mammalian sperm by imaging capabilities will undoubtedly impact the study of using fluorophore-conjugated phalloidin. To compare
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